Metal ions play important roles in many biological systems. Cells utilize metal ions for a wide variety of functions, such as regulating enzyme activities, protein structures, cellular signaling, as catalysts, as templates for polymer formation and as regulatory elements for gene transcription. Metal ions can also have a deleterious effect when present in excess of bodily requirements or capacity to excrete. A large number of natural and synthetic materials are known to selectively or non-selectively bind to or chelate metal ions. Ion chelators are commonly used in solution for in vivo control of ionic concentrations and detoxification of excess metals, and as in vitro buffers. Ion chelators can be used as optical indicators of ions when bound to a fluorophore, and may be useful in the analysis of cellular microenvironments or dynamic properties of proteins, membranes and nucleic acids. For example, Ca2+ ions play an important role in many biological events, and so the determination of intracellular Ca2+ is an important biological application.
Fluorescent indicators utilizing a polycarboxylate BAPTA chelator have been predominantly used for intracellular calcium detections (see U.S. Pat. No. 4,603,209; U.S. Pat. No. 5,049,673; U.S. Pat. No. 4,849,362; U.S. Pat. No. 5,453,517; U.S. Pat. No. 5,501,980; U.S. Pat. No. 5,459,276; U.S. Pat. No. 5,501,980; U.S. Pat. No. 5,459,276; and U.S. Pat. No. 5,516,911; each of which is hereby incorporated by reference). Xanthene-based fluorescent calcium indicators (such as Fluo-3, Fluo-4 and Rhod-) are the most common fluorescent indicators used in biological assays. However, these existing xanthene-based calcium indicators typically have low fluorescence quantum yields, resulting in low detection sensitivity. Furthermore their corresponding acetoxymethyl esters may not readily penetrate the membranes of live cells (thus requiring higher temperatures to achieve optimal dye loading), and once inside the cells, they exhibit a slow conversion to the corresponding BAPTA free acid.
In view of the existing drawbacks for currently used xanthene-based fluorescent calcium indicators, what is needed are improved compositions and methods that offer sensitive detection of small variations in calcium concentrations, with a rapid response and a strong fluorescence signal. Also needed are fluorescent indicators that can be readily loaded into live cells. In addition, compositions and methods that are less susceptible to the effects of external changes (such as temperature) are preferred for high throughput screening and high content analysis.
The present application is directed to a family of fluorescent dyes that are useful for preparing fluorescent metal ion indicators. The indicators include a fluorescein lactone fluorophore and an ionophore, and are useful for the detection, discrimination and quantification of metal cations. The fluorescent indicators of this invention demonstrate unexpected better cellular retention compared to the existing fluorescein ion indicators.